Estrogen is a major risk factor for breast cancer. The biological effects of estrogen are mediated primarily through the two estrogen receptors (ER), ER-alpha and ER-beta. The hypothesis addressed in this proposal is that alterations in components of estrogen receptor signaling pathways occur during breast tumorigenesis, and that certain etiologic subsets of breast tumors will be defined by these alterations. The proposed study will evaluate several new markers related to estrogen signaling and responsiveness in the Carolina Breast Cancer Study (CBCS), a large population-based, case-control study of invasive and in situ breast cancer in African-American and white women. In a small pilot study, we have detected the ER-alpha (A to G at nucleotide 908) mutation recently reported by Fuqua et al (2000) at relatively high levels in invasive and in situ breast cancers. For the proposed study, we will carry out more comprehensive laboratory analyses to detect the ER-alpha A908G mutation in invasive and CIS breast tumors, assess its mRNA expression in an allele-specific manner, and localize the mutation to histologic subregions of tumors with both invasive and CIS components. We will evaluate the immunohistochemical expression of ER-beta, ER-alpha, and the combined expression of ER-alpha and ER-beta in invasive and CIS breast tumors. Finally, we will screen the germline of invasive cancer cases and controls for CYP19/aromatase genotypes. Using statistical analyses, we will: (1) Determine the prevalence of the ER-alpha A908G mutation, and expression of ER-beta and/or ER-alpha in invasive and CIS tumors, and the relationship among these markers; (2) Examine correlations between the ER markers and breast tumor histologic or clinicopathologic characteristics as well as previously-identified somatic alterations, including p53 mutation or overexpression, HER-2/neu gene amplification or overexpression, and expression of Ki-67, E-cadherin and VEGF; (3) Examine the correlation of hormonal risk factors or CYP19/aromatase or CYP17 genotypic variation with breast tumor subsets defined by each somatic ER marker; and (4) Assess the risk of ER-defined breast cancer subtypes associated with hormonal risk factors. As treatment and follow-up information becomes available for the CBCS cases, we also plan to assess the clinical significance of these ER markers with response to hormonal therapies and survival.